LOCUS       sequence-472503-        7525 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses H. vulgare RbcL RbcS-6xHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7525)
  AUTHORS   Archer J, Kathpalia M, Lee BU, Li S, Wang T
  TITLE     Effects of chaperone selectivity on the assembly of plant Rubisco 
            orthologs in E. coli.
  JOURNAL   J Exp Bot. 2025 Mar 31:eraf140. doi: 10.1093/jxb/eraf140.
  PUBMED    40163664
REFERENCE   2  (bases 1 to 7525)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Exp Bot. 
            2025 Mar 31:eraf140. doi: 10.1093/jxb/eraf140."
COMMENT     Sequence Label: pTW335v3
FEATURES             Location/Qualifiers
     source          1..7525
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     170..189
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     423..440
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    455..594
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(520..539)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     680..702
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(696..887)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2199..3281)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3243..3262
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3282..3359)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(3602..3607,3608..3623,3624..3630)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3638..3654
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3643..3665
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     RBS             5184..5206
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5604..5621
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5703..5750
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5984..6002)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6070..6174
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6175..6243,6244..7035)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6393..6412)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7206..7525,1..269)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
       61 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      121 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
      181 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
      241 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
      301 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
      361 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
      421 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
      481 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
      541 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
      601 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
      661 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
      721 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
      781 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
      841 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
      901 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
      961 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     1021 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     1081 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     1141 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     1201 accattcatg ttgttgctca ggtcgcagat gttttgcagc agcagtcgct tcacgttcgc
     1261 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     1321 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
     1381 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
     1441 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
     1501 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     1561 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
     1621 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
     1681 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
     1741 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     1801 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     1861 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     1921 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     1981 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     2041 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     2101 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     2161 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2221 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2281 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     2341 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     2401 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     2461 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     2521 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     2581 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     2641 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     2701 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     2761 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     2821 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     2881 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     2941 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     3001 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     3061 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     3121 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     3181 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     3241 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     3301 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcg
     3361 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac aattctcatg tttgacagct
     3421 tatcatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
     3481 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
     3541 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
     3601 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
     3661 acaggaaaca gccagtccgt ttaggtgttt tcacgagcac ttcaccaaca ataggtgcag
     3721 taaggagata taccatgcca caaacagaga ctaaagcggg tgttgggttc caagctggtg
     3781 ttaaagatta taaattgact tactatactc ctgaatatga aaccaaggat actgatatct
     3841 tggcagcatt ccgagtatcg cctcaacctg gagttccacc tgaagaagca ggggctgcgg
     3901 tagctgctga atcttctact ggtacatgga caactgtgtg gaccgatggg cttaccagcc
     3961 ttgatcgtta caaaggacga tgctaccaca tcgagcccgt tgcgggggag gatagtcaat
     4021 ggatctgtta tgtagcttat cccttagacc tttttgaaga aggttcggtt actaacatgt
     4081 ttacctcgat tgtgggtaat gtatttgggt tcaaagccct gcgcgctcta cgtctagagg
     4141 atctgcgaat ccctcctacc tacagtaaaa ctttccaagg accacctcat ggtatccaag
     4201 ttgaaagaga taaattgaac aagtatggac gtcccctatt aggatgtact attaaaccaa
     4261 aattggggtt atccgcgaaa aactatggta gagcatgtta tgaatgtcta cgtggtggac
     4321 ttgattttac caaagatgat gagaatgtga actcccaacc atttatgcgt tggagagatc
     4381 gtttcgtctt ttgtgccgaa gctatttata aatcacaggc tgaaacaggt gaaatcaaag
     4441 ggcattattt gaatgctact gcgggtacat gcgaagaaat gatcaaaaga gctgtatttg
     4501 ccagagaatt gggagttcct atcgtaatgc atgactactt aacaggggga ttcaccgcaa
     4561 atactacgtt ggcgcattat tgccgagata atggcctact tcttcacatc caccgtgcaa
     4621 tgcacgctgt tattgataga cagaagaatc atggtatgca cttccgtgta ctagctaaag
     4681 ctttacgtat gtctggtgga gatcatattc actcgggtac agtagtaggt aaacttgaag
     4741 gagaacgcga aatgactttg ggctttgttg atttactgcg cgatgatttt attgagaaag
     4801 atcgtgcgcg cggtatcttt ttcactcaag attgggtttc aatgccgggc gtcatacctg
     4861 tggcttcagg gggtattcac gtttggcaca tgcctgcttt gaccgagatc tttggagatg
     4921 attctgtact acaattcggt ggaggaactt taggccaccc ttggggaaat gcaccgggtg
     4981 ccgcggccaa ccgagtagct ctggaagcat gtgtacaagc tcgtaatgag ggacgtgatc
     5041 ttgcacgtga gggtaatgaa attatccgtg cggcttgcaa atggagtcct gaactagctg
     5101 ctgcttgtga agtatggaaa gcaattaaat tcgaatttga gccggtcgat acgattgaca
     5161 aaaaggtgta agctagaaat aattttgttt aactttaaga aggagatata catatgtgta
     5221 tgcaagtttg gcccattgaa gggattaaaa aatttgaaac cttatcatac ctcccgcctc
     5281 tttccactga agccctgctg aaacaggtgg attatctgat ccgtagcaaa tgggtgccgt
     5341 gcctggagtt ttcgaaagtg ggctttatct tccgtgaaca taatgcaagc ccgggatatt
     5401 acgatggtcg gtactggacg atgtggaagc tgccaatgtt tggttgcacc gatgccacac
     5461 aggtgttgaa cgaagttgaa gaggtaaaaa aagaatatcc cgacgcgtat gtccgcataa
     5521 ttggcttcga caacatgcgc caggttcaat gtgtctcttt catcgcgttt aaaccgccgg
     5581 gctgtcaaga gagtggtaag gctcatcacc accatcatca ttaacatccg gctgctaaca
     5641 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataaacc gctgagcaat
     5701 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     5761 gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc
     5821 tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca
     5881 cggtgcctga ctgcgttagc aatttaactg tgataaacta cagcattaaa gctagcttat
     5941 cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta
     6001 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     6061 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6121 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6181 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6241 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6301 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6361 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     6421 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6481 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6541 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6601 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6661 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     6721 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6781 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6841 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg atctcgcggt
     6901 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6961 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     7021 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     7081 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7141 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7201 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7261 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7321 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     7381 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7441 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7501 gataaggcgc agcggtcggg ctgaa
//
